WO2004029262A2

WO2004029262A2 - Production of 2 - keto - l - gulonic acd

Info

Publication number: WO2004029262A2
Application number: PCT/EP2003/010490
Authority: WO
Inventors: Tatsuo Hoshino; Teruhide Sugisawa; Yoshinori Takagi
Original assignee: Dsm Ip Assets B.V.
Priority date: 2002-09-27
Filing date: 2003-09-22
Publication date: 2004-04-08
Also published as: AU2003270233A8; WO2004029262A3; CN100506995C; CN1681933A; AU2003270233A1

Abstract

The present invention relates to a process for the production of 2-KGA or a salt thereof from D-sorbitol by a multi-stage continuous or a single-stage semi-continuous fermentation mode using a mixed culture of microorganisms of the genus Gluconobacter or Acetobacter. One of the microorganisms is G. oxydans DSM 4025.

Description

Production of 2-KGA

The present invention relates to a process of fermentative production of 2-keto-L- gulonic acid (2-KGA) from D-sorbitol in high yield by utilizing the fermentation system composed of the multi-stage continuous mode or single-stage semi-continuous (repeated fed-batch) mode.

2-KGA is an important intermediate for the production of L-ascorbic acid. This compound can be converted to L-ascorbic acid according to the well-known Reichstein method. Japanese Patent Publication No.4.0154/ 1976 discloses the production of 2-KGA from D-sorbitol by microorganisms of the genus Acetobacter or Pseudomonas. These microorganisms are capable of oxidizing D-sorbitol under aerobic condition producing 2-KGA, however, with a low yield of less than 6 g/L. Due to the relatively low yields, these processes are far from an industrial scale of production, especially when D-sorbitol is the starting material.

Various Acetobacter (presently classified into Gluconobacter) strains such as A xylinum and A. suboxydans are known to efficiently produce L-sorbose from D-sorbitol. D-sorbitol is a less costly material than L-sorbose, but L-sorbose is more efficiently converted to 2-KGA.

The present invention provides a process for producing 2-KGA from D-sorbitol in high yield by either a multi-stage continuous fermentation or by a single-stage semi- continuous (repeated fed-batch) fermentation.

More particularly, the present invention relates to a process for the production of

2-keto-L-gulonic acid (2-KGA) or a salt thereof from D-sorbitol by a multi-stage continuous or a single-stage semi-continuous fermentation using a mixed culture of G. oxydans DSM 4025 (FERM BP-3812) or a mutant thereof having the identifying characteristics of G. oxydans DSM 4025 (FERM BP-3812), and a microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L- sorbose from D-sorbitol, and, optionally, recovering the 2-KGA from the fermentation broth.

G. oxydans DSM 4025 was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) in Gδttingen (Germany), based on the stipulations of the Budapest Treaty, under DSM No. 4025 on March 17, 1987. The depositor was The Oriental Scientific Instruments Import and Export Corporation for Institute of Microbiology, Academia Sinica, 52 San-Li-He Rd., Beijing, Peoples Republic of China. The effective depositor was said Institute, of which the full address is The Institute of Microbiology, Academy of Sciences of China, Haidian, Zhongguancun, Beijing 100080, People's Republic of China.

A subculture of the strain has also been deposited at the National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan, also based on the stipulations of the Budapest Treaty, under the deposit No. FERM BP-3812 on March 30, 1992. The depositor is Nippon Roche K.K., 6-1, Shiba 2-chome, Minato-ku, Tokyo 105-8532 Japan. This subculture is also most preferably used in the present invention.

The multi-stage continuous fermentation comprises more than one fermentation vessels. An example of the multi-stage continuous system is the two-stage continuous fermentation system, which consists of a medium reservoir, a 1^st fermentor, a 2ⁿ fermentor, and a harvesting vessel. By using more than one fermentation vessel in the entire process, the yield of the process is optimized.

In the conventional batch or fed-batch operations, biomass is discarded after each batch resulting in higher production cost and the time required for reactor clean-up and start-up (inoculation) resulting in the loss of reactor productivity.

The multi-stage continuous culture mode of the 2-KGA fermentation process results in higher productivity and concentrations of 2-KGA without leaving residual D- sorbitol and L-sorbose. This is a preferred method for the isolation step in preparing sodium 2-KGA.

In the start-up procedure of the continuous fermentation a batch or a fed-batch operation can be applied.

In the present invention, it is possible to produce 2-KGA from D-sorbitol without immobilization methods for microorganisms such as chemical bonding, or physical methods for cell retention, e.g., matrix entrapment, and without biomass hold back or retention such as membrane system. Therefore, when the multi-stage fermentative production system of 2-KGA from D-sorbitol is constructed, the mixed cultivation of G. oxydans DSM 4025 and a microorganism capable of producing L-sorbose from D- sorbitol can be easily and directly applied as a low-cost 2-KGA fermentation process. The D-sorbitol concentration of the initial production medium and the feeding medium for continuous mode can be about 0 to about 250 g/L for semi-continuous mode and 5 to 250 g/L for multi stage continuous mode, respectively. Additionally, the feeding medium for semi-continuous mode can contain D-sorbitol in the range between about 20 and about 800 g/L. The feeding volume for fed-batch mode may vary depending on the working volume and the fermentation conditions. In the range of the D-sorbitol concentration of the feeding medium for continuous mode, the dilution rate can be set at about 0.01 to about 0.05 h^"1.

The present invention provides a process for the production of 2-KGA or a salt thereof from D-sorbitol by continuous fermentation, comprising:

(a) cultivating, in one or more fermentation vessel, a microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L-sorbose from D- sorbitol together with G. oxydans DSM 4025 (FERM BP-3812) or a mutant thereof having the identifying characteristics of G. oxydans DSM 4025 (FERM BP-3812), in a nutrient medium containing D-sorbitol;

(b) continuously feeding a nutrient medium containing D-sorbitol to the fermentation vessel(s) in a concentration of about 5 to about 250 g/L, with the dilution rate of the fermentation set at about 0.01 to about 0.05 h^" ;

(c) continuously withdrawing fermentation broth from the fermentation vessel(s); and (d) recovering 2-KGA from the fermentation broth.

The present invention also relates to the single-stage semi- continuous fermentation process to produce 2-KGA from D-sorbitol. The semi- continuous fermentation, in which a part of the culture broth is used as the seed for the next fermentation, is one of the methods to achieve high 2-KGA productivity from D-sorbitol. About 10% (v/v) of whole culture broth after 48 h cultivation may be used as the seed for the 2ⁿ step, and the fermentor is filled with the fresh nutrient medium, and cultivated. Repeating this cycle can be operated, successively. In contrast to the conventional batch or fed-batch operations, the semi- continuous operation can utilize the cultivation broth as the seed for the next cultivation. Therefore, the operation can be applied for the fermentation of 2-KGA without several seed steps from the small scale seed and the time for the preparation of the culture such as the clean-up and start-up time. Furthermore, a higher reactor productivity can be kept for a long fermentation period.

Thus, it is an object of the present invention to provide a process for the production of 2-KGA or a salt thereof from D-sorbitol by semi-continuous fermentation, comprising:

(a) cultivating in a fermentation vessel a microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L-sorbose from D-sorbitol together with G. oxydans DSM 4025 (FERM BP-3812) or a mutant thereof having the identifying characteristics of G. oxydans DSM 4025 (FERM BP-3812), in a nutrient medium containing D-sorbitol in an initial concentration of about 0 to about 250 g/L, and the feeding medium for fed-batch mode containing D-sorbitol in a concentration of about 20 to about 800 g/L,

(b) using a part of the fermentation broth as the seed cultivation for the next fed-batch fermentation,

(c) successively repeating this procedure, and

(d) recovering 2-KGA from the fermentation broth.

The volume ratio of the seed to the next fermentation may be in the range between about 0.1 and about 90% (v/v), preferably in the range between about 5 and about 90%

(v/v).

The period of one fed-batch fermentation can be varied depending on the volume of the seed for the next fermentation cycle or fermentation conditions such as the D- sorbitol concentration of the initial production medium and the feeding medium for fed- batch mode.

Therefore, in the present invention, the period of one fed-batch fermentation cycle can be harmonized with the amount of the seed for the next fermentation in order to enhance the 2-KGA productivity.

The D-sorbitol concentration of the initial production medium may be about 0 to about 250 g/L. Additionally, the feeding medium for fed-batch mode can contain D- sorbitol in the range between about 20 and about 800 g/L.

The continuous and semi- continuous fermentations ma be carried out at a dissolved oxygen concentration in the fermentation broth from about 0.1 to about 100% air saturation, preferably between about 5 to about 100% air saturation.

Thus, it is an aspect of the present invention to provide a process for the production of 2-KGA or a salt thereof form D-sorbitol by a multi-stage continuous or a single-stage semi-continuous fermentation using a mixed culture of G. oxydans DSM 4025 (FERM BP-3812) or a mutant thereof having the identifying characteristics of G. oxydans DSM 4025 (FERM BP-3812), and a microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L-sorbose from D-sorbitol, wherein the fermentation is carried out at the dissolved oxygen concentration in the fermentation broth between about 0.1 and about 100%. In a preferred embodiment, the dissolved oxygen concentration is between about 5 and about 100%. In addition to D-sorbitol used as starting material in the present invention, other substances which are carbon sources may also be added, such as glycerol, D-glucose, D- mannitol, D-fructose, D-arabitol and the like.

Various organic or inorganic substances may also be used as nitrogen sources, such as yeast extract, meat extract, peptone, casein, corn steep liquor, urea, amino acids, nitrates, ammonium salts and the like. As inorganic substances, magnesium sulfate, potassium phosphate, ferrous and ferric chlorides, calcium carbonate and the like may be used.

The mixing ratio of these nutrients and amounts of each ingredient may vary with the properties of the microorganisms employed. The amounts of the starting material, D-sorbitol, the amount of one of the microorganisms to be inoculated with respect to the other and the times of inoculations, and the other conditions of the incubation maybe selected or determined in accordance with the particulars of the individual case.

The conditions of the cultivation may also vary depending on the species and character of the microorganisms employed. The composition of the medium may, of course, be selected or determined in accordance with the particulars of the individual case in order to yield the intended product most efficiently, although cultivation temperatures between about 13°C and about 36°C, preferably between about 18°C and about 33°C, and a pH value of the medium between about 4.0 and about 9.0, preferably between about 6.0 and about 8.0, may be maintained.

Thus, in one embodiment of the present invention the process for the production of 2-KGA or a salt thereof from D-sorbitol by a multi-stage continuous or a single-stage semi- continuous fermentation as above, wherein the fermentation is carried out at a pH between about 4.0 and about 9.0. A preferred pH is between about 6.0 and about 8.0.

A further embodiment of the present invention is the process for the production of

2-KGA or a salt thereof from D-sorbitol by a multi-stage continuous or a single-stage semi-continuous fermentation as above, wherein the fermentation is carried out at a temperature between about 13°C and about 36°C. A preferred embodiment is a temperature between about 18°C and 33°C.

In order to maintain the pH value of the medium, any suitable acidic or basic reagent may be added to the medium in a suitable amount at a suitable time during the cultivation. The same object may alternatively be accomplished by incorporating a suitable buffer or buffering of the cultivation. L-Sorbose produced from D-sorbitol by the L-sorbose producing strain can be used by the 2-KGA producing strain to form 2-KGA. Any microorganism capable of converting D-sorbitol to L-sorbose maybe used in this invention in combination with G. oxydans DSM 4025.

Examples of microorganisms capable of producing L-sorbose from D-sorbitol belonging to the genus Gluconobacter or Acetobacter include G. suboxydans IFO 3130, G. suboxydans IFO 3255, G. suboxydans IFO 3256, G. suboxydans IFO 3257, G. suboxydans IFO 3258, G. suboxydans IFO 3289, G. suboxydans IFO 3290, G. suboxydans IFO 3291, G. gluconicus IFO 3171, G. gluconicus IFO 3285, G. gluconicus IFO 3286, G. gluconicus IFO 3244, G. albidus IFO 3251, G. albϊdus IFO 3253, G. industrius IFO 3261, G. cerinus IFO 3262, G. cerinus IFO 3263, G. cerinus IFO 3265, G. cerinus IFO 3266, G. cerinus IFO 3267, G. cerinus IFO 3270, G. diacetonicus IFO 3273, G. røseus IFO 3990, A. αceri spp. Orleans IFO 3259, A. aceti spp. αcetz IFO 3281, A. Uquefaciens IFO 12257, A. Uquefaciens IFO 12258, A. liquefacience IFO 12388, A cet. spp. xylinum IFO 3288, A. ceft- spp. xylinum IFO 13693, A. cef. spp. xylinum IFO 13772 and A αcet. spp. xylinum IFO 13773.

Thus, the present invention relates to a process for the production of 2-KGA or a salt thereof from D-sorbitol by a multi-stage continuous or a single-stage semi- continuous fermentation using a mixed culture of G. oxydans DSM 4025 (FERM BP- 3812) or a mutant thereof having the identifying characteristics of G. oxydans DSM 4025 (FERM BP-3812), and a microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L-sorbose from D-sorbitol, wherein said microorganism is selected from the group consisting of Gluconobacter suboxydans, Gluconobacter rubiginosus, Gluconobacter albidus, Gluconobacter industrius, Gluconobacter cerinus, Gluconobacter diacetonicus, Gluconobacter roseus, Gluconobacter gluconicus, Acetobacter aceti spp. Orleans, Acetobacter aceti spp. xylinum, and Acetobacter Uquefaciens. Preferred microorganisms are selected from the group consisting of G. suboxydans IFO 3255, G. suboxydans IFO 3256, G. suboxydans IFO 3258, G. suboxydans IFO 3290, G. suboxydans IFO 3291, G. gluconicus IFO 3285, and G. cerinus IFO 3267. More preferably, the microorganism the fermentation is carried out by the mixed cultivation of G. suboxydans IFO 3291 and G. oxydans DSM 4025.

The above microorganisms are preserved in public depositories for delivery to anyone upon request. One such depository is the Institute of Fermentation, Osaka, Japan (IFO).

The microorganisms as defined above also include synonyms or basonyms of such species having the same physico-chemical properties, as defined by the International Code of Nomenclature of Prokaryotes. The microorganisms are usually cultured in an aqueous medium containing D- sorbitol and appropriate nutrients such as nitrogen sources and minor elements, under aerobic conditions. However, any conventional fermentation condition can be utilized in carrying out the process of this invention.

The 2-KGA obtained according to the present process can be isolated from the reaction mixture, e.g., by the formation of a salt or by using differences in properties between the product and the surrounding impurities, such as solubility, absorbability and distribution coefficient between the solvents. Adsorption, e.g., on ion exchange resins constitutes a convenient means for isolating the product. The thus obtained product may further be purified in a conventional manner, e.g., by recrystallization or chromatography. Alternatively, the reaction mixture can be used directly for conversion to L-ascorbic acid by esterification, followed by enolization and lactonization.

The present invention is illustrated by the following Examples. Conversion yield is calculated based on the amounts of 2-KGA produced against D-sorbitol used.

Example 1: Two-stage 2-KGA continuous fermentation from 12% D-sorbitol by the mixed cultivation of G. oxydans DSM 4025 and G. suboxydans IFO 3291 with DO control

(1) Preparation of seed culture for strain G. oxydans DSM 4025

A seed culture medium containing 8% L-sorbose (separately sterilized), 0.05% glycerol, 0.25% MgSO₄ ^«7H₂0, 1.75% corn steep liquor, 5.0% baker's yeast (pH 7.0 before sterilization), 0.5% CaCO₃, 0.5% urea (separately sterilized) and 0.03% antifoam (Actcol: Takeda) was distributed into a 500 ml Erlenmeyer flask (100 ml each) and sterilized at 121°C for 20 minutes. Into this seed culture medium, one loopful amount of the cells of microorganism DSM 4025 was inoculated and incubated for 24 h at 30°C.

(2) Preparation of seed culture for G. suboxydans IFO 3291

A seed culture medium containing 2% D-sorbitol, 0.3% yeast extract (Oriental Yeast), 0.3% beef extract, 0.3% corn steep liquor, 1% polypeptone, 0.1% urea, 0.1% KH₂PO₄, 0.02% MgSO₄-7H₂O, 0.1% CaCO₃ (pH 7.0 before sterilization) and 0.03% antifoam was sterilized by autoclaving at 121°C for 20 minutes. One loopful amount of the cells of G. suboxydans IFO 3291 were transferred into 100 ml of the seed culture medium in a 500 ml Erlenmeyer flask and incubated for 24 h at 28°C. (3) Inoculation to the main fermentation

100 ml of each seed culture was prepared (total seed volume was 200 ml) and inoculated into 2 L of the initial production medium containing 4%D-sorbitol, 1% corn steep liquor, 0.01% MgSO₄-7H₂O, 0.025% KH₂PO₄, 0.4% yeast extract (BYF 100: Universal Foods) and 0.15% antifoam. After the 2-KGA concentration increased up to 120 g/L by fed-batch mode, which phase was corresponding to 90% of DO (dissolved oxygen) level, the culture mode was switched to the continuous mode.

(4) Two-stage continuous fermentation

In the two-stage continuous fermentation system the glass jar fermentor was equipped with the same apparatus as described above. This fermentation system consisted of two fermentors. Both fermentors had a working volume of 2 L. In order to enhance the 2-KGA productivity and the dilution rate for the continuous fermentation, the fed-batch operation in the start-up procedure for the continuous fermentation was applied. The medium for the fed-batch operation was composed of 60% D-sorbitol, 6.67% corn steep liquor, 0.0287% MgSO₄-7H₂O, 0.0717% KH₂PO₄ and 0.2% antifoam. During the 30 h fed-batch operation, 600 ml of the feeding medium for the fed-batch mode was supplied to the main fermentor with a first peristaltic pump. After that, the continuous feeding rate was controlled with a second peristaltic pump. The broth withdrawn from the 1^st fermentor was further transferred to the 2ⁿ stage fermentor with a 3^r peristaltic pump. Finally, the broth was withdrawn to the harvest reservoir with a 4 peristaltic pump while the working volume was kept at 2 L. The temperature was controlled at 28°C and the agitation speed and aeration rate were set at 800 rpm and 1.0 L/min, respectively. The pH was controlled at 7.0 with sodium hydroxide solution.

It was known that the DO control for the 2-KGA production from D-sorbitol by this mixed cultivation was effective to enhance the G. suboxydans IFO 3291 cell activity and 2-KGA production. Therefore, DO level in the 1^st stage fermentor was controlled at 10% during hours 9 to 52 from the beginning of the fermentation. DO level was controlled by supplying the air mixed with pure oxygen. D (dilution rate) was set in the range of 0.035 to 0.045 h^"1 in both of the 1^st and 2^nd fermentors. The fermentation was continued for 250 h. Table 1 shows the fermentation activity of the mixture of G. oxydans DSM 4025 and G. suboxydans IFO 3291 by this two-stage continuous mode. In the continuous mode, 74.5 g/L of the average 2-KGA was produced. The productivity of 2-KGA in the 1^st fermentor was calculated to be 2.57 g/L/h at the steady state of the continuous mode. Moreover, the overall productivity was calculated to be 1.33 g/L/h and the overall 2-KGA molar conversion yield was 60.6%. Table 1: Fermentation activity in the two-stage continuous mode using 3 L fermentor

Example 2: Two-stage 2-KGA continuous fermentation from 13% D-sorbitol by the mixed cultivation of G. oxydans DSM 4025 and G. suboxydans IFO 3291

The initial production medium and the feeding medium for the fed-batch mode were composed of the same ingredients as described in Example 1. The two-stage continuous fermentation was operated as described in Example 1. The two-stage 2-KGA continuous fermentation used the continuous feeding medium (10 L) containing 13% D- sorbitol, 3% corn steep liquor, 0.25% MgSO ^«7H₂O, 0.05% glycerol, yeast extract (BYF100) and 0.15% antifoam.

The fermentation was carried out for 160 h. The relatively stable fermentation activity was kept for the fermentation period and the average D was 0.0361 h^" in both of the 1^st and 2ⁿ fermentors. The results are summarized in Table 2. In the steady state of the continuous mode, 88.2 g/L of 2-KGA was produced. The productivity of 2-KGA in the 1^st fermentor was calculated to be 2.28 g/L/h at the steady state of the continuous mode. The overall productivity was calculated to be 1.59 g/L/h with the molar conversion yield of 63.0% from D-sorbitol. 2-KGA productivity using this continuous feeding medium was at least 19% higher than that described in Example 1.

Table 2: Fermentation activity in the two-stage continuous mode using a 3 L fermentor

Example 3: Two-stage 2-KGA continuous fermentation from 14% D-sorbitol by the mixed cultivation of G. oxydans DSM 4025 and G. suboxydans IFO 3291

In this two-stage continuous fermentation, D-sorbitol concentration in the continuous feeding medium was 14%. Other components in the continuous feeding medium were the same as in Example 2. The two-stage continuous fermentation was operated as described in Example 1. The initial production medium and the feeding medium for the fed-batch mode (600 ml) were the same as the media described in Example 1.

D was varied in the range of 0.015 to 0.060 h^"1 and 0.020 to 0.050 h^"1 for the 1^st and 2 _.n"d^u . fermentors, respectively, at appropriate times to investigate the D-value necessary to convert D-sorbitol to 2-KGA completely without D-sorbitol and L-sorbose left in the final harvest tank. The fermentation was stopped at the 700 hour. The fermentation activity of the mixture of G. oxydans DSM 4025 and G. suboxydans IFO 3291 by the two- stage continuous mode is summarized in Table 3. The broth containing 75.1 g/L of 2- KGA was obtained at an apparent D of 0.0331 h^"1 in the two-stage continuous mode, and the overall dilution rate was calculated to be 0.0166 h^"1. The overall 2-KGA molar conversion yield was 48.9%. The productivity of 2-KGA in the 1^st fermentor and the overall productivity of 2-KGA were calculated to be 2.07 and 1.24 g/L/h at the steady state of the continuous mode, respectively. This two-stage continuous method used 10% DO control in the 1^st fermentor. The DO control in the 1^st fermentor could enhance the G. suboxydans IFO 3291 cell activity of converting D-sorbitol to L-sorbose, and by-products such as 2-KD (2-keto-D-gluconic acid)were not accumulated in the culture broth in the final fermentor. A more steady 2-KGA production could be carried out by using the DO control.

Table 3: Fermentation activity in the two-stage continuous mode using a 3 L fermentor

Example 4: Single-stage 2-KGA semi-continuous fermentation from D-sorbitol by the mixed cultivation of G. oxydans DSM 4025 and G. suboxydans IFO 3291

100 ml of each seed culture as described in Example 1 was inoculated into 1.5 L of the initial production medium containing 2% D-sorbitol, 3% corn steep liquor, 0.25% MgSO₄-7H₂O, 0.05% glycerol, 0.4% BYF100 and 0.15% antifoam. The composition of the feeding medium for the fed-batch mode (550 ml) consisted of 60% D-sorbitol, 10.67% corn steep liquor, 0.023% MgSO₄-7H₂O, 0.0573% KH₂PO and 0.2% antifoam. The 2-KGA semi-continuous production medium was composed of the same ingredients as the initial production medium. The amount of D-sorbitol in the initial medium and the feeding medium per one batch was applied 30 g and 330 g, respectively. The interval time of one fed-batch fermentation was set at 30 h. Upon complete consumption of the initial D-sorbitol, the fed-batch fermentation mode was started.

After 30 h of the 1^st fed-batch fermentation 90% (v/v) of the culture broth was harvested. The remaining 10% (v/v) of the culture broth left in the fermentor was used as the seed for the next fermentation. The fermentor was filled with the semi- continuous production medium containing the same ingredients as the initial production medium.

After that, 5% (v/v) of G. suboxydans IFO 3291 were inoculated into the main fermentor.

By repeating this cycle successively, the fermentation in the main fermentor could continuously be repeated. The pH was controlled at 7.0 with NaOH during the fermentation and 10% of DO control was applied for the semi- continuous fermentation.

In the 2-KGA semi-continuous fermentation from D-sorbitol, the temperature was controlled at 28°C. The agitation speed and aeration rate were set at 800 rpm and 0.75

L/min, respectively.

The main fermentation was repeated 5 times and the total fermentation period was

165 h. The total amount of D-sorbitol per one batch operation in both the initial and the feeding medium was 360 g. Table 4 shows the 2-KGA fermentation activity in the semi- continuous mode. The 2-KGA production was in the range of between 119.5 and 130.7 g/L and the average 2-KGA production was 124.6 g/L. The 2-KGA productivity per one batch was in the range of between 3.96 and 4.33 g/L/h and the average 2-KGA productivity was 4.14 g/L/h. The molar conversion yield from D-sorbitol was more than 70.0%. This semi- continuous 2-KGA fermentation was significantly stable and this process could produce more than 119 g/L of 2-KGA and easily maintain more than 4.00 g/L/h of the average 2-KGA productivity during the long production period. Table 4: Fermentation activity in the single-stage semi- continuous mode using a 3 L fermentor

Example 5: Effect of the shorter interval time of one batch on the 2-KGA single-stage semi-continuous fermentation from D-sorbitol using a new production medium

The interval time of one fed-batch fermentation was set at 24 h. The initial and semi- continuous production medium used was the same medium as described in Example 4. The amount of D-sorbitol in the initial medium and the feeding medium per one batch was 30 g and 330 g, respectively.

The main fermentation was repeated 5 times and the total fermentation period was 130 h. The total amount of D-sorbitol per one batch operation in both the initial and the feeding medium was 360 g. Table 5 shows the 2-KGA fermentation activity in the semi- continuous mode for the shorter interval time per one batch. The 2-KGA production was in the range of between 105.6 and 110.8 g/L and the average 2-KGA production was 108.2 g/L. The 2-KGA productivity per one batch was in the range between 4.40 and 4.62 g/L/h and the average 2-KGA productivity was 4.51 g/L/h. The productivity was higher by 8.94% than that described in Example 4. The molar conversion yield from D-sorbitol was more than 61.0%. The 24 h of interval time could enhance the average 2-KGA productivity in the semi-continuous operation.

Table 5: Fermentation activity in the single-stage semi- continuous mode using a 3 L fermentor

Claims

1. A process for the production of 2-keto-L-gulonic acid (2-KGA) or a salt thereof from D-sorbitol by a multi-stage continuous or a single-stage semi- continuous fermentation using a mixed culture of G. oxydans DSM 4025 (FERM BP-3812) or a mutant thereof having the identifying characteristics of G. oxydans DSM 4025 (FERM BP-3812), and a microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L-sorbose from D-sorbitol, and, optionally, recovering the 2-KGA from the fermentation broth.

2. The process according to claim 1, wherein the multi-stage continuous fermentation comprises the steps of:

(b) continuously feeding a nutrient medium containing D-sorbitol to the fermentation vessel(s) in a concentration of about 5 to about 250 g/L, with the dilution rate of the fermentation set at about 0.01 to about 0.05 h^"1;

3. The process according to claim 1, wherein the semi-continuous fermentation comprises the steps of:

(a) cultivating in a fermentation vessel a microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L-sorbose from D-sorbitol together with G. oxydans DSM 4025 (FERM BP-3812) or a mutant thereof having the identifying characteristics of G. oxydans DSM 4025 (FERM BP-3812), in a nutrient medium containing D-sorbitol in an initial concentration of about 0 to about 250 g/L, and the feeding medium for fed-batch mode containing D-sorbitol in a concentration of about 20 to about 800 g/L, (b) using a part of the fermentation broth as the seed cultivation for the next fed-batch fermentation,

(c) successively repeating this procedure, and

(d) recovering 2-KGA from the fermentation broth.

4. The process according to claim 3, wherein the volume ratio of the seed cultivation to the next fed-batch fermentation is in the range between about 0.1 and about 90% v/v, preferably in the range between about 5 and about 90% v/v.

5. The process according to claim 1, wherein the fermentation is carried out at the dissolved oxygen concentration in the fermentation broth between about 0.1 and about 100%, preferably between about 5 and about 100%.

6. The process according to claim 1, wherein the fermentation is carried out at a pH between about 4.0 and about 9.0, preferably between about 6.0 and about 8.0.

7. The process according to claim 1, wherein the fermentation is carried out at a temperature between about 13°C and 36°C, preferably between about 18°C and 33°C.

8. The process according to claim 1, wherein the microorganism capable of converting D-sorbitol to L-sorbose is a microorganism selected from the group consisting of Gluconobacter suboxydans, Gluconobacter rubiginosus, Gluconobacter albidus, Gluconobacter industrius, Gluconobacter cerinus, Gluconobacter diacetonicus, Gluconobacter roseus, Gluconobacter gluconicus, Acetobacter aceti spp. Orleans, Acetobacter aceti spp. xylinum, and Acetobacter Uquefaciens.

9. The process according to claim 8, wherein the microorganism is selected from the group consisting of G. suboxydans IFO 3255, G. suboxydans IFO 3256, G. suboxydans IFO 3258, G. suboxydans IFO 3290, G. suboxydans IFO 3291, G. gluconicus IFO 3285, and G. cerinus IFO 3267.

10. The process according to claim 1, wherein the fermentation is carried out by the mixed cultivation of G. suboxydans IFO 3291 and G. oxydans DSM 4025.